Project Summary/Abstract Morphine and most clinically used opioid analgesics, as well as heroin, act primarily through mu opioid receptors. The single-copy mu opioid receptor gene (OPRM1) undergoes extensive alternative pre-mRNA splicing, generating an array of splice variants that are conserved from rodents to humans. One type of the splice variants are full-length 7-transmembrane (TM) C-terminal variants that are identical except for the sequences at the tip of the intracellular C-terminal tail. Increasing evidence supports the pharmacological importance of these 7TM C-terminal variants. Several in vitro cell models demonstrate functional differences in mu agonist-induced G protein coupling, phosphorylation, internalization and post-endocytic sorting, as well as region- and cell-specific expression. More importantly, in vivo functions of several C-terminal variants were recently revealed in C-terminal truncation mouse models with two inbred mouse background. Particularly, exon 7 (E7)-associated C-terminal truncation in C57BL/6J strain diminished morphine tolerance and reward without altering physical dependence, whereas the E4-associated C-terminal truncation accelerated morphine tolerance and reduced morphine dependence without affecting morphine reward. Together, these studies underscore the functional importance of these C-terminal splice variants in mediating the diverse actions of mu opioids, and provide a compelling rationale to further explore molecular mechanisms of C-terminal 7TM splice variants in mu opioid actions, as proposed in this application. We hypothesize that different C-terminal sequences of the Oprm1 full-length 7TM variants are important in determining interaction of a receptor with a unique set of proteins either at basal states or in response to mu agonists, leading to their distinct signaling pathways and functions. In this application we propose using newly developed proximity-dependent biotin identification with an engineered ascorbate peroxidase (APEX2) coupled with tandem mass tag (TMT) proteomics approach, to map these transient or dynamic receptor-protein interactions under both basal state and activated conditions in response to different mu agonists in OPRM1-KD Be(2)C cells and primary striatal neurons derived from Oprm1 knockout mice. We will compare two E7-associated C-terminal 7TM variants mMOR-1O and mMOR-1C that have unique in vitro and in vivo pharmacological profiles with E4-associated mMOR-1, and a truncated version, mE1/2/3, that lacks additional C-terminal sequences downstream of E3. We will validate physical and/or functional interactions of selected candidates from APEX2-TMT study using a number of approaches, such as NanoLuc Binary Technology (NanoBit) and RNAi. The proposed studies promise to reveal new insights into mu agonist-induced receptor-protein interactions, signaling and function of the C-terminal splice variants, and to provide a general approach applicable to all G-protein coupled receptors. With approximately 12% of non-olfactory GPCRs having alternative C-terminal splice variants, the results from this application may have a very broad impact.